Broad spectrum vaccine against gonorrhea

ABSTRACT

Vaccines comprising peptide sequences corresponding to immunorecessive determinants in gonorrhea pilus protein are disclosed. The vaccines are effective in protecting human subjects against infection by a wide range of gonorrhea strains by raising antibodies which interfere with the colonization of the epithelium by the infecting bacteria or which enhance phagocytosis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending U.S. patentapplication Ser. No. 623,178, filed 21 June 21, 1984, now U.S. Pat. No.4,584,195.

TECHNICAL FIELD

This invention relates to immunization of humans against gonorrhealinfection. In particular, it relates to vaccines useful in protectinghumans against a broad spectrum of gonococcus strains.

BACKGROUND ART

Gonorrhea is a well known, sexually transmitted disease which producesacute suppuration of the mucous membranes of the genital urinary tractand the eye followed by chronic inflammation and fibrosis. It is causedby a gram negative group of cocci Neisseria gonorrhoeae (gonococcus). Asingle strain of this species is an isolate from a single host (patient)at a particular site. There are, therefore, multitudinous strains of N.gonorrhoeae, each of which has characteristic antigenic determinantsassocated with the pili, a fact which renders both diagnosis andimmunization difficult. The incidence of the disease has markedlyincreased since 1955, and has been complicated by the appearance ofpenicillin resistant strains harboring β-lactamase encoding plasmids,which were first reported in 1976. The infectivity of the organism isextremely high, and it has been estimated that a single sexual encounterwith an infected partner results in a 20-30% probability of acquiringthe disease. If left untreated, relapses are to be expected, asresistance to re-infection does not appear to develop.

The course of the disease involves colonization of the mucous membranesby the bacterium, a process which is mediated by the attachment of thecolonizing cell to the surface membrane by means of filamentousstructures called pili associated with its cell wall. After attachment,the gonococcus is passed through the epithelium to the submucosal spacewhere it is capable of causing inflammation and fibrosis. The attachmentof the gonococci to the epithelial surface can be blocked by anti-pilusantibody.

In addition to blocking cell attachment, antibodies raised against pilusprotein are also opsonic--i.e., they mediate the killing of the invadingbacteria by the phagocytes in the blood. However, the use of pilusimmunogens as vaccines has been rendered impractical by the lack ofcross-reactivity among strains.

The lack of cross-reactivity of antibodies raised against pili ofvarious strains has been shown not to be absolute. (Brinton, C. C. Jr.et al, Immunobiology of Neisseria Gonorrheae (1978) American Society forMicrobiology, Washington, D.C., p. 155). Also, the nature of the pilusprotein has been studied. The filamentous portion consists of apolymeric form of a monomeric polypeptide, pilin, and the complete aminoacid sequence of the pilin isolated from the transparent colonialvariant (Tr) of strain MS11 and a partial sequence of R10 (Tr) pilinhave been determined (Schoolnik, G. K. et al, J. Exp. Med. (1984)159:1351). It has also been shown that when the pilin associated witheither of these two strains is treated with cyanogen bromide, twoimmunologically important fragments CNBr-2, residues 9-92, and CNBr-3,residues 93-159, are generated which appear to represent immunologicallydifferent portions of the molecule. CNBr-3 is apparently antigenicallyvariable and immunodominant; CNBr-2 apparently contains a conservedreceptor-binding region and is immunorecessive. (Schoolnik, G. K. et al,Prog. Allergy (1983) 33:314). None of the foregoing work has resulted ina material which can serve as a effective vaccine against all strains ofN. gonorrhoeae. By providing an immunogenic form of an antigenicdeterminant capable of eliciting antibodies reactive against all strainseither by inhibiting attachment, or by encouraging phagocytosis or both,protection would be provided against gonnorheal infection. This is theaccomplishment of the present invention.

DISCLOSURE OF THE INVENTION

It has now been found that certain portions of the amino acid sequenceassociated with the pilus protein are capable, when made immunogenic, ofeliciting antibodies which are capable not only of reacting against piliof a wide spectrum of gonococcal strains, but also of either preventingthe adherence of these strains to epithelial cells or aiding theirdestruction by phagocytes, or both. Some of the peptide sequences arethose associated with the immunorecessive, receptor-binding portion ofthe conserved region of the pilin, and reside in the CNBr-2 fragment.Others reside in the CNBr-3 portion. When these antigenically reactivepeptide sequences are bound to a carrier protein so as to conferimmunogenicity upon them, they can be used as a vaccine to protect aperson injected or otherwise administered the vaccine against gonorrhea.Gonorrhea appears to infect only human beings and not other mammals, andthus an animal model of the disease does not exist, nor is there a needfor a vaccine to protect other animals against the disease.

Accordingly, in one aspect, this invention relates to a vaccineeffective against gonorrhea infection in humans which comprises aneffectively protective amount of a peptide of an antigenic, conservedsequence substantially equivalent to the amino acid sequence representedby residues 21-35, 41-50, 48-60, 69-84, 107-121, 135-151 or 151-159 ofN. gonorrhoeae MS11(Tr) linked to a substantially antigenically neutralcarrier protein.

In other aspects, the invention relates to these amino acid sequenceswith additional residues at the carboxy termini to facilitate linkage tothe carrier protein in the correct orientation, and to the conjugationproducts resulting. It also, in other aspects, relates to the foregoingpeptide sequences in substantially pure form, and to methods forprotecting human beings against gonorrheal infection using the vaccinesof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of N-methyl(phenylala) MS11 pilin,and the regions representing the proteins of the invention.

MODES OF CARRYING OUT THE INVENTION A. Definitions

As used herein, "substantially equivalent to" in characterizing apeptide sequence means that the substantial equivalent is capable ofcarrying out the same antigenic function and mediating the immunologicfunction of the referenced sequence. In general, the sequence of aminoacids in the substantially equivalent peptide and the referenced peptidewill be identical, however, as is well understood, it may be possible tosubstitute or modify a small number of these residues withoutappreciable impact on the performance of the resulting polypeptide.Means are now understood in the art for deleting, adding, or modifyingindividual amino acid residues either directly or by altering theircoding sequences, and modifications so performed which result inpolypeptide sequences of equivalent performance generate peptides whichare "substantially equivalent".

"Peptide", "polypeptide", and "protein" are used interchangeably, andrefer to amino acid sequences of a variety of lengths, either in theirneutral (uncharged) forms or in forms which are salts, and either freeof modifications such as glycosylation, side chain oxidation, orphosphorylation or containing these modifications. It is well understoodin the art that amino acid sequences contain acidic and basic groups,and that the particular ionization state exhibited by the peptide isdependent on the pH of the surrounding medium when the protein is insolution, or that of the medium from which it was obtained if theprotein is in solid form. Also included in the definition are proteinsmodified by additional substituents attached to the amino acid sidechains, such as glycosyl units, lipids, or inorganic ions such asphosphates, as well as modifications relating to chemical conversions ofthe chains, such as oxidation of sulfhydryl groups. Thus, "peptide" orits equivalent terms is intended to include the appropriate amino acidsequence referenced, subject to those of the foregoing modificationswhich do not destroy its functionality.

"Substantially antigenically neutral carrier" refers to a material towhich the peptides of the invention may be attached to render themimmunogenic, but which does not itself elicit antibodies which will bedetrimental to the host, or contain antigenic sites which interfere withthe antigenic function of the invention peptides. In the illustrationbelow, as rabbits were used as a source of antibody, bovine serumalbumin (BSA) could be used. For human use, however, carriers would belimited to proteins which do not raise antibodies to materials commonlyand non-pathogenically encountered by humans. For example, the somatic"Protein I" of the gonococcus itself could be used, as could tetanustoxoid protein. The use of other carriers is not precluded; however,these are the most convenient forms of serologically compatible carriersand are, at the present time, the most conveniently used representativesof this class.

B. General Description

B.1. Gonococal Pili and the Antigenic Sequences

Pilin isolated from N. gonorrhoeae strain MS11(Tr) has been shown to bea 159 amino acid peptide having two cysteine residues, at positions 121and 151, which are joined by a disulfide link to create a loop in thechain between these two positions. (See FIG. 1.) The presence ofmethionine residues at positions 7 and 92 gives rise to three cyanogenbromide fragments two of which have been used extensively inimmunological studies; i.e., CNBr-1 (residues 1-7); CNBr-2 (residues8-92) and CNBr3 (residues 93-159 ). The amino acid sequence of CNBr-2contains two domains represented by approximately positions 41-50 and69-84, which are hydrophilic and which can be shown by computer analysisof the sequence to contain beta turns, thus indicating their proximityto the surface of the protein. The amino acid sequences in these regionsrepresent two of the immunogenic portions of the vaccines of theinvention which generate adherence-blocking antibodies. The antibodiesgenerated in response to these positions are also opsonic. In addition,peptides substantially equivalent to positions 48-60 and 135-151function, when linked to carriers, are immunogens capable of raisinghigh-titer opsonic antibodies; peptides substantially equivalent topositions 21-35, 107-121 and 151-159 also raise opsonic antibodieshaving somewhat lower titers. The seven peptides capable of raisingopsonic antibodies are indicated in FIG. 1.

B.2. Linkers

Because the peptide sequences above are considered too small to beimmunogenic, they have been linked to carrier substances in order toconfer this property upon them. Any method of creating such linkagesknown in the art may be used.

Linkages can be formed in a variety of ways. For example, there are alarge number of heterobifunctional agents which generate a disulfidelink at one functional group end and a peptide link at the other, andthese have been used extensively. The most popular of these isN-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP). This reagentcreates a disulfide linkage between itself and a cysteine residue in oneprotein and an amide linkage through the ε amino on a lysine, or otherfree amino group in the other. A variety of such disulfide/amide formingagents are known. See for example Immun. Rev. (1982) 62:185. Otherbifunctional coupling agents form a thioether rather than a disulfidelinkage. Many of these thioether forming agents are commerciallyavailable and include reactive esters of 6-maleimidocaproic acid, 2bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like. The carboxyl groups can beactivated by combining them with succinimide or1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. The particularlypreferred coupling agent for the method of this invention issuccinimmidyl 4-(N-maleimido-methyl) cyclohexane-1-carobxylate (SMCC)obtained from Pierce Company, Rockford, Illinois. The foregoing list isnot meant to be exhaustive, and modifications of the named compounds canclearly be used.

B.3. Methods of Preparation of the Immunogenic Active Ingredient

The antigenic peptides of the invention can be prepared in a number ofconventional ways. Because they are short sequences, they can beprepared by chemical synthesis using standard means. Particularlyconvenient are solid phase techniques (see for example Erikson, B. W. etal, The Proteins (1976) v. 2, Academic Press, New York, p. 255). Indeed,automated solid phase synthesizers are commerically available, as arethe reagents required for their use. Thus, not only is it possible tomimic the sequence of amino acids in the designated positions of theMS11 pilin, modifications in the sequence can easily be made bysubstitution, addition or omission of appropriate residues. Particularlyconvenient modifications, as set forth above, include the addition of acysteine residue at the carboxy terminus to provide a sulfhydryl groupfor convenient linkage to the carrier protein. In addition, spacerelements, such as an additional glycine residue may be incorporated intothe sequence between the linking amino acid at the C-terminus and theremainder of the peptide.

Also because the desired sequences are relatively short, recombinanttechniques to produce these peptides are particularly appealing. Thecoding sequence for peptides of this length can easily be synthesized bychemical techniques, for example, the phosphotriester method ofMatteucci, M. et al, J Am Chem Soc (1981) 103:3185. Of course, bychemically synthesizing the coding sequence, any desired modificationcan be made simply by substituting the appropriate bases for thoseencoding the native peptide sequence. The coding sequence can then beprovided with appropriate linkers and ligated into expression vectorsnow commonly available in the art, and the resulting vectors used totransform suitable hosts to produce the desired protein.

A number of such vectors and suitable host systems are now available.For example promoter sequences compatible with bacterial hosts areprovided in plasmids containing convenient restriction sites forinsertion of the desired coding sequence. Typical of such vectorplasmids are, for example, pUC8, and pUC13 available from Messing, J.,at the University of Minnesota; (see, e.g., Messing, et al, NucleicAcids Res (1981) 9:309) or pBR322, available from New England Biolabs.Suitable promoters include, for example, the β-lactamase (penicillinase)and lactose (lac) promoter systems (Chang, et al, Nature (1977) 198:1056and the tryptophan (trp) promoter system (Goeddel, D., et al, NucleicAcids Rec (1980) 8:4057)). The resulting expression vectors aretransformed into suitable bacterial hosts using the calcium chloridemethod described by Cohen, S. N., et al, Proc Natl Acad Sci USA (1972)69:2110. Successful transformants may produce the desired polypeptidefragments at higher levels than those found in strains normallyproducing the intact pili. Of course, yeast or mammalian cell hosts mayalso be used, employing suitable vectors and control sequences.

Finally, these sequences can also be prepared by isolation of nativepilin and hydrolysis of the peptides obtained. However, this method isthe least practical, as large amounts of unwanted protein must beeliminated from the original preparation.

The antigenic peptide sequence containing suitable modification toprovide for linkage can then be provided with a suitable antigenicallyneutral carrier using any of a variety of linking agents as set forthabove. Suitable carriers include the aforementioned Protein I andtetanus toxoid and these can be linked to the antigenic peptide through,for example, bifunctional linkers providing thioether or disulfide linksand amide linkages. The conditions of such linkage of course depend onthe nature of the linker used, and are well understood in the art.

B.4. Vaccine Preparation

Prepration of vaccines which contain peptide sequences as activeingredients is also well understood in the art. Typically, such vaccinesare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinjection may also be prepared. The preparation may also be emulsified.The active immunogenic ingredient is often mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccine. Thevaccines are conventionally administered parenterally, by injection, forexample, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of manitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

The proteins may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, capacity of the subject's immune system to synthesizeantibodies, and the degree of protection desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges are of the order of several hundred micrograms activeingredient per individual. Suitable regimes for initial administrationand booster shots are also variable, but are typified by an initialadministration followed in one or two week intervals by a subsequentinjection or other administration.

C. EXAMPLES

The following are intended to illustrate but not to limit the invention.

C.1. Preparation of Active Ingredients

The peptide substantially equivalent to positions 41-50,Glu-Gly-Gln-Lys-Ser-Ala-Val-Thr-Glu-Tyr, was synthesized on a commercialBeckman Model 990B Peptide Synthesizer using commerically availableamino acid polystyrene resins and t-Boc protected amino acids (PeninsulaLaboratories, Belmont, California), with the following side chainprotecting groups: O-benzyl esters for Asp, Glu, Thr, and Ser; tosyl forArg and His; p-methoxybenzyl for Cys, o-chlorobenzyloxycarbonyl for Lys,and 2, 6-dichlorobenzyl for Tyr. Coupling was performed using a 2.5molar excess of t-Boc amino acid and dicyclohexylcarbodiimide (DCC) overresin bound amino acid. In the case of Asn and Gln, a 2.5 molar excessof the amino acid, DCC, and N-hydroxytriazole was used. All couplingswere more than 99% complete, as determined by the reaction of the resinwith ninhydrin. The peptides were simultaneously deprotected and removedfrom the resin by treatment with anhydrous HF in the presence ofanisole, dimethylsulfide, and indole. The peptides were separated fromthe various organic side products by extraction with ether, isolatedfrom the resin by raction with 5% acetic acid and then lyophilized. Thepurity of the crude product was determined by HPLC on a C-18 reversephase column (Merck, Darmstadt, Germany) and by amino acid analysis. Thepeptide was determined to be more than 90% pure.

In a similar manner the following peptides were prepared:

For additional peptides representing positions 41-50:

Glu-Gly-Gln-Lys-Ser-Ala-Val-Thr-Glu-Tyr-Cys;

Glu-Gly-Gln-Lys-Ser-Ala-Val-Thr-Glu-Tyr-Gly-Cys.

For positions 21-35

Leu-Pro-Ala-Tyr-Gln-Asp-Tyr-Thr-Ala-Arg-Ala-Gln-Val-Ser-Glu;

Leu-Pro-Ala-Tyr-Gln-Asp-Tyr-Thr-Ala-Arg-Ala-Gln-Val-Ser-Glu-Cys; and

Leu-Pro-Ala-Tyr-Gln-Asp-Tyr-Thr-Ala-Arg-Ala-Gln-Val-Ser-Glu-Gly-Cys.

For positions 48-60:

Thr-Glu-Tyr-Tyr-Leu-Asn-His-Gly-Lys-Trp-Pro-Glu-Asn;

Thr-Glu-Tyr-Tyr-Leu-Asn-His-Gly-Lys-Trp-Pro-Glu-Asn-Cys; and

Thr-Glu-Tyr-Tyr-Leu-Asn-His-Gly-Lys-Trp-Pro-Glu-Asn-Gly-Cys.

For positions 69-84:

Pro-Pro-Ser-Asp-Ile-Lys-Gly-Lys-Tyr-Val-Lys-Glu-Val-Glu-Val-Lys;

Pro-Pro-Ser-Asp-Ile-Lys-Gly-Lys-Tyr-Val-Lys-Glu-Val-Glu-Val-Lys-Cys; and

Pro-Pro-Ser-Asp-Ile-Lys-Gly-Lys-Tyr-Val-Lys-Glu-Val-Glu-Val-Lys-Gly-Cys.

For positions 107-121:

Ser-Leu-Trp-Ala-Arg-Arg-Glu-Asn-Gly-Ser-Val-Lys-Trp-Phe-Cys; and

For positions 135-151:

Asp-Ala-Lys-Asp-Gly-Lys-Glu-Ile-Asp-Thr-Lys-His-Leu-Pro-Ser-Thr-Cys;

For positions 151-159:

Cys-Arg-Asp-Lys-Ala-Sar-Asp-Ala-Lys;

Arg-Asp-Lys-Ala-Ser-Asp-Ala-Lys-Cys; and

Arg-Asp-Lys-Ala-Ser-Asp-Ala-Lys-Gly-Cys.

C.2. Linkage to Carrier Protein

The peptides from paragraph C.1. which contain a C-N-terminal Cysresidue are linked to bovine serum albumin using succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (Pierce,Rockford, Ill.) as described by Yoshitake, S. et al, Eur J. Biochem.(1979) 101: 395. Briefly, 10 mg BSA is dissolved in 2 ml phosphatebuffered saline (PBS), pH 7.4 and combined with 5 mg of SMCC in 0.5 mlof dimethylformamide. After one hour at room temperature, the conjugatewas separated from unreacted SMCC by gel filtration on G-25 in 0.1 Mphosphate, pH 6.0.

The peptide Glu-Gly-Gln-Lys-Ser-Ala-Val-Thr-Glu-Tyr-Cys was dissolved in0.1M borate, pH 9.1 and reduced with NaBH₄ (0.1 ml of 0.1M stock). Afterfive minutes, the pH of the borate solution was lowered to 1 with 1M HClto remove excess NaBH₄ and then raised to pH 6 with 1M NaOH, andcombined with the linker-BSA conjugate. After incubating at roomtemperature for an additional hour, the peptide-linker-BSA conjugate wasdesalted on a G-25 column in 0.1M NH₄ HCO₃. The degree of conjugationwas quantitated by comparing the amino acid composition of the BSAbefore and after reaction with the peptide. The conjugate containedapproximately 15-25 peptides per molecule of BSA.

In a similar manner, the other C-terminal Cys containing peptides ofparagraph C.1. were conjugated with BSA.

C.3. Confirmation of Antigenicity of Peptide Conjugates

Several procedures were employed to confirm that the peptide conjugatesrepresenting positions 41-50 and 69-84 had the ability to bindspecifically to antibodies against gonococcal pili. The antibodiestested were raised against purified whole pili derived from R10 or MS11or against CNBr2 and CNBr3 fragments thereof. The antibodies were raisedin rabbits using standard techniques and were therefore polyclonalpreparations. Assessment was done using RIA, ELISA, and by adsorption bypeptide-carrier-sepharose conjugates.

For RIA and ELISA, 96 well plates were coated with 10 mg of the peptideBSA conjugate to be tested, washed, treated with serially dilutedantiserum, washed, and then treated with reagent. For RIA, the reagentwas 125I protein A (Amersham, Arlington Heights, Ill.). For ELISA, thereagent was goat anti-rabbit alkaline phosphatase conjugate (Cappel,Westchester, PA) followed by p-nitrophenyl phosphate. Radioactive wells(RIA) were cut from the plate and counted. For the ELISA the wells wereread at 405 nm.

In the sepharose adsorption assay, the peptide-BSA conjugates werereacted with CNBr activated sepharose (Pharmacia, Piscataway, NJ) asdescribed by Porath, J. et al, Meth Enzymol (1974) 34: 13. The antibodycontaining serum (1 ml) was exposed with 0.1 ml of thepeptide-carrier-sepharose for 2 hours at room temperature, and themixture separated by centrifugation. Supernatant was then assayed forthe presence of antibody using a standard solid phase binding assay.

These assays showed a mixed response. The only anti-sera to which thepeptide conjugates prepared in paragraph C.2 bound were those raisedagainst the CNBr2 fragment of MS11 or R10. Conjugates containingantigenic peptides substantially similar both to protein 41-50 and to69-84 bound to anti R10 CNBr2, only the 69-84 conjugate bound well toanti-MS11-CNBr2. Neither was responsive to antibodies against wholepili.

These results are consistent with the immunorescessive nature of theseantigenic determinants. It would, indeed, be expected that immunogenicregions having the potential for generating antibodies of high crossreactivity would, in the context of species generating highly strainspecific antibodies, be recessive.

C.4. Confirmation of Adherence Blocking Ability.

To test the ability of antibodies raised against the conjugated peptidesof the invention to block adherence to target cells, an in vitro assaywas used.

To obtain the target cells, epithelial cells derived from humanendometrial cells were grown in monolayer tissue culture on cover slips.

An inoculum of piliated gonococcal strain F62 was pre-incubated withvarying serum dilutions and transferred to a chamber containing thecultured cells. After 30 minutes of incubation, unbound bacteria wereremoved by repeated washings. The cover slip was strained using Giemsaand the number of adhering bacteria counted.

The results are shown in Table 1. "Pre" refers to a control serum, i.e.,without immunization with the indicated peptide. "Post" refers to serumobtained from immunized animals.

To obtain the "post" immune serum, the peptide to be tested wasconjugated to carrier and prepared as a vaccine in complete Freunds'sadjuvant as follows: 500 μg of the conjugate in phosphate bufferedsaline (PBS) was emulsified with an equal volume of adjuvant. Thevaccine was administered to 7 kg female New Zealand white rabbits byintramuscular or subcutaneous injection. Boosters were given after 6weeks with similar vaccine and the animals were bled 10 days thereafterand the sera prepared for assay.

                                      TABLE 1                                     __________________________________________________________________________    Antisera Against:                                                             Serum 41-50 Conjugate                                                                          48-60 Conjugate                                                                           69-84 Conjugate                                  Dilution.sup.-1                                                                     Pre   Post Pre   Post  Pre    Post                                      __________________________________________________________________________    10    37.2 + 7.8*                                                                         28 + 1.6                                                                           48.3 + 14.0                                                                         55.8 + 15.3                                                                         26.3 + 9.4                                                                            1.6 + 1.5                                25    64.9 + 14.5                                                                         9.2 + 3.9                                                                          .sup. N.D..sup.+                                                                    N.D.  47.5 + 10.1                                                                           7.6 + 2.6                                50    82.5 + 9.1                                                                          9.4 + 4.6                                                                          N.D.  N.D.  170.0 + 21.2                                                                         10.2 + 3.9                                100   N.D.  N.D. N.D.  N.D.  93.7 + 11.3                                                                          10.8 + 2.2                                __________________________________________________________________________     *Bacteria per cell ± S.D.                                                  .sup.+ N.D.  Not determined.                                             

The results in Table 1 show that immune serum against the peptideconjugate substantially equivalent to MS11 pilin positions 69-84 waseffective in blocking adherence to epithelial cells at all dilutionstested (at least 1:100), and that the antiserum raised against pilinpositions 41-50 was comparably effective. A control using a peptideconjugate corresponding to positions 48-60 showed no ability to inhibitbinding.

In summary, vaccines prepared from conjugates of peptides substantiallyequivalent to positions 41-50 or 69-84 of MS11 pili are protectiveagainst infection by virtue of their ability to raise antibodies whichinhibit adherence of gonoccal pili derived from a broad spectrum ofstrains.

C.5. Confirmation of Opsonic Activity for Antibodies Generated byConjugates of Invention Peptides

Antibodies were raised as set forth in C.4 by injecting the conjugatesinto rabbits. The resulting antisera were tested for opsonic behaviorusing a modification of the method of Cohn, Z. A., et al, J Exp Med(1959) 110:419.

Human polymorphonuclear leukocytes (PMN's) were prepared from a 10 mlsample of heparinized blood. The red cells were sedimented in phosphatebuffered saline containing 5% dextran, and the leukocyte richsupernatant removed and washed in heparin-saline by repeatedcentrifugation. The concentrated leukocytes were then suspended ingelatine-Hanks' salt solution and their concentration adjusted byhemocytometer counting to 1-2×10⁷ PMN's/ml.

Piliated phase variant gonococci, heterologous strain F62, was grown onsolid typing media for 18 hr at 6.5° C. in 5.6% CO₂ and air, thebacteria were harvested with a bent glass rod into Hanks' salt solutionand gelatine, and the cell concentration adjusted by nephelometry to1-2×10⁷ microorganisms/ml.

Serial dilutions of anti-sera prepared against the conjugates wereheat-inactivated (56° C.×45 min), serially diluted in Hanks' saltsolution and gelatine, and combined with equal volumes of the PMN's andbacteria. The reaction was allowed to proceed at 37° C. under continuousrotation (4 rev/min). After an incubation period of 15 min, phagocytosiswas stopped instantly by bringing the reactants to 2° C. Thebacteria-cell suspension was washed by centrifugation in Hanks' solutionwith gelatine, the cell pellet resuspended in Hanks' solution with 10%serum and the reactants were incubated at 37° C. At 0, 30, 60, 90 and120 min aliquots were removed and intracellular killing stopped by theaddition of ice-cold Hanks' solution. The PMN's were then harvested bycentrifugation and lysed by the addition of ice-cold distilled watercontaining 0.1% BSA. Serial ten-fold dilutions in saline were used todetermine the number of viable bacteria.

The results are shown in Table 2 as percent of surviving bacteria at 120min compared to a control sample containing pre-immune antisera.

                                      TABLE 2                                     __________________________________________________________________________    Bactericidal Activity of Anti-Peptide Sera                                    and Human Polymorphonuclear Leukocytes                                        Surviving Bacteria/Control Survivors × 100                              Anti-Sera Against Conjugates with                                             Serum Constant Region Peptides                                                                      Variable Region Peptides                                Dilution                                                                            21-35                                                                             41-50                                                                             48-60                                                                             69-84                                                                             107-121                                                                            121-134                                                                            135-151                                                                            151-159                                  __________________________________________________________________________    1:10  15.6                                                                               8.6                                                                               5.1                                                                               4.6                                                                              12.4 78-1 22-4 48.8                                     1:25  33.8                                                                              14.8                                                                              11.3                                                                               8.2                                                                              28.9 94.6 32.6 63.2                                     1:50  98.6                                                                              34.2                                                                              28.7                                                                              18.6                                                                              86.4 100  48.8 89.1                                     1:100 100 81.3                                                                              62.8                                                                              58.4                                                                              100  100  69.1 100                                      1:200 100 100 88.1                                                                              76.3                                                                              100  100  88.2 100                                      __________________________________________________________________________

The results show anti-sera raised against conjugates of peptidessubstantially equivalent to positions 41-50, 48-60, 69-84, and 135-151to have high titers of opsonic activity; those raised against conjugatessubstantially equivalent to positions 21-35, 107-121, and 151-159 havelower titers but are opsonic; that raised against conjugatessubstantially equivalent to positions 121-134 had little opsonicactivity.

We claim
 1. A vaccine protective against gonorrhea which comprises aneffectively protective amount of a peptide wherein the only antigenicdeterminant is an amino acid sequence that corresponds to residues21-35, 48-60, 107-121, 135-151 or 151-159 inclusive of N. gonorrhoeaeMS11(Tr) pilin linked to a substantially antigenically neutral carrier.2. The vaccine of claim 1 wherein the amino acid sequence whichcorresponds to residues 21-35, or 48-60 or to residues 151-159, whereinthe N-terminal Cys is deleted from the amino acid sequence whichcorresponds ro residues 151-159, further contains a Cys residue or aGly-Cys dipiptide at its C terminus.
 3. A vaccine protective againstgonorrhea which comprises an effectively protective amount of a peptidewherein the only antigenic determinant is an amino acid sequenceselected from the group consisting of(a)Leu-Pro-Ala-Tyr-Gln-Asp-Tyr-Thr-Ala-Arg-Ala-Gln-Val-Ser-Glu; (b)Thr-Glu-Tyr-Tyr-Leu-Asn-His-Gly-Lys-Trp-Pro-Glu-Asn; (c)Ser-Leu-Trp-Ala-Arg-Arg-Glu-Asn-Gly-Ser-Val-Lys-Trp-Phe-Cys; (d)Asp-Als-Lys-Asp-Gly-Lys-Glu-Ile-Asp-Thr-Lys-His-Leu-Pro-Ser-Thr-Cys; and(e) Cys-Arg-Asp-Lys-Ala-Ser-Asp-Ala-Lyssaid peptide linked tosubstantially antigenically neutral carrier.
 4. The vaccine of claim 3,wherein each sequency of a or b, or of e, wherein the N-terminal Cys isdeleted from e, further includes a C-terminal Cys residue or C-terminalGly-Cys residues.
 5. The vaccine of claim 3, wherein the peptide hassequence (a).
 6. The vaccine of claim 3, wherein the peptide hassequence (b).
 7. The vaccine of claim 3, wherein the peptide hassequence (c).
 8. The vaccine of claim 3, wherein the peptide hassequence (d).
 9. The vaccine of claim 3, wherein the peptide hassequence (e).
 10. A peptide in substantially pure form which comprisesas the sole antigenic determinant an amino acid sequence selected fromthe group consisting of(a)Leu-Pro-Ala-Tyr-Gln-Asp-Tyr-Thr-Ala-Arg-Ala-Gln-Val-Ser-Glu; (b)Thr-Glu-Tyr-Tyr-Leu-Asn-His-Gly-Lys-Trp-Pro-Gly-Asn; (c)Ser-Leu-Trp-Ala-Arg-Arg-Glu-Asn-Gly-Ser-Val-Lys-Trp-Phe-Cys; (d)Asp-Ala-Lys-Asp-Gly-Lys-Glu-Ile-Asp-Thr-Lys-His-Leu-Pro-Ser-Thr-Cys; and(e) Cys-Arg-Asp-Lys-Ala-Ser-Asp-Ala-Lys.
 11. The peptide of claim 10,wherein each sequence of a or b, or of e wherein the N-terminal Cys isdeleted from e, further includes a C-terminal Cys residue or C-terminalGly-Cys residue.
 12. The peptide of claim 10, which has sequence (a).13. The peptide of claim 10, which has sequence (b).
 14. The peptide ofclaim 10, which has sequence (c).
 15. The peptide of claim 10, which hassequence (d).
 16. The peptide of claim 10, which has sequence (e).